Bottom Line:
Furthermore, we find that the normal rate of droplet initiation depends on 14 amino acids at the amino terminus of seipin, deletion of which results in fewer, larger droplets that are consistent with a delay in initiation but are otherwise normal in morphology.Importantly, other functions of seipin, namely vectorial budding and resistance to inositol, are retained in this mutant.We conclude that seipin has dissectible roles in both promoting early LD initiation and in regulating LD morphology, supporting its importance in LD biogenesis.

Figure 1: Seipin knockout drastically impedes de novo droplet formation. A galactose-inducible promoter was integrated into the genomic DGA1 locus in a strain lacking the other three NL acyltransferases (3KO(GALDGA1)) and in a strain with an additional seipin knockout (3KO(GALDGA1)fld1∆). Cells were introduced to rich galactose medium at t = 0 to induce droplet formation and stained with BODIPY at the indicated time points. (A) Representative fluorescence microscopy projection images at indicated time points after galactose induction, overlaid onto bright-field images. Scale bar: 5 μm. (B) Image of typical cells from each strain at 3 h. Scale bar: 2 μm. Intensity settings were kept constant between the two images to compare membrane brightness. (C) Percent of cells containing at least one FB. Error bars signify range from two experiments. (D) Number of distinct FBs per cells that have at least one FB. Error bars represent SDs from two experiments. (E) TG levels determined by TLC of lipid extracts from whole-cell lysates, normalized to cell pellet wet weight. Error bars represent range from 2 independent experiments. (F) TG synthesis activity of isolated membranes. Error bars signify SDs from two experiments. (G) Growth of cultures. Starting OD was 0.3. Error bars, SEM from two experiments, each performed in duplicate.

Mentions:
Cells were analyzed at 0, 3, 6, and 9 h after the switch to galactose. Micrographs at 0 h revealed no BODIPY staining, except for a single droplet in a rare 3KO(GALDGA1) cell, while 82% of cells in this strain produced droplets after only 3 h; this number increased to 98% by 9 h after induction (Figure 1, A–C). By 9 h, cells typically contained approximately five droplets (Figure 1D).

Figure 1: Seipin knockout drastically impedes de novo droplet formation. A galactose-inducible promoter was integrated into the genomic DGA1 locus in a strain lacking the other three NL acyltransferases (3KO(GALDGA1)) and in a strain with an additional seipin knockout (3KO(GALDGA1)fld1∆). Cells were introduced to rich galactose medium at t = 0 to induce droplet formation and stained with BODIPY at the indicated time points. (A) Representative fluorescence microscopy projection images at indicated time points after galactose induction, overlaid onto bright-field images. Scale bar: 5 μm. (B) Image of typical cells from each strain at 3 h. Scale bar: 2 μm. Intensity settings were kept constant between the two images to compare membrane brightness. (C) Percent of cells containing at least one FB. Error bars signify range from two experiments. (D) Number of distinct FBs per cells that have at least one FB. Error bars represent SDs from two experiments. (E) TG levels determined by TLC of lipid extracts from whole-cell lysates, normalized to cell pellet wet weight. Error bars represent range from 2 independent experiments. (F) TG synthesis activity of isolated membranes. Error bars signify SDs from two experiments. (G) Growth of cultures. Starting OD was 0.3. Error bars, SEM from two experiments, each performed in duplicate.

Mentions:
Cells were analyzed at 0, 3, 6, and 9 h after the switch to galactose. Micrographs at 0 h revealed no BODIPY staining, except for a single droplet in a rare 3KO(GALDGA1) cell, while 82% of cells in this strain produced droplets after only 3 h; this number increased to 98% by 9 h after induction (Figure 1, A–C). By 9 h, cells typically contained approximately five droplets (Figure 1D).

Bottom Line:
Furthermore, we find that the normal rate of droplet initiation depends on 14 amino acids at the amino terminus of seipin, deletion of which results in fewer, larger droplets that are consistent with a delay in initiation but are otherwise normal in morphology.Importantly, other functions of seipin, namely vectorial budding and resistance to inositol, are retained in this mutant.We conclude that seipin has dissectible roles in both promoting early LD initiation and in regulating LD morphology, supporting its importance in LD biogenesis.